Currently, renewable fatty acids are obtained solely from plant oils. Medium chain fatty acids (C8-C14) are typically sourced from coconut and palm oil, whereas longer chain saturated and unsaturated fatty acids are typically sourced from tallow, soy, corn or sunflower oil. Fatty acids are widely used for food, personal care products, industrial applications (e.g., lubricants, adhesives, detergents and plastics), as well as increasingly as biofuels. The demand for renewable fatty acids is rising and expanding.
With the current understanding of biological pathways it becomes possible to utilize other organisms, especially microorganisms, for the production of renewable chemicals such as fatty acids. Microbial fatty acid synthesis proceeds via a stepwise addition of 2 carbon units onto a growing acyl chain bound to acyl carrier protein (ACP). The process begins as a condensation of acetyl-ACP and malonyl-ACP into acetoacetyl-ACP liberating CO2 which drives the reaction forward. The second step involves reduction of acetoacetyl-ACP to D-3-hydroxybutyryl-ACP using NADPH. Following a dehydration to crotonyl-ACP and another reduction using NADPH, butyryl-ACP is formed. The chain elongation typically continues with further addition of malonyl-ACP until a C16 acyl chain is formed, which is then hydrolyzed by a thioesterase into a free C16 fatty acid.
Although many organisms show preference for fatty acid termination at C16 carbon chain length, various plant thioesterases show a broad range of specificity terminating at both shorter and longer chain lengths. Moreover, some thioesterases are capable of terminating monounsaturated fatty acids such as C16:1 (C16 refers to fatty acid with a 16 carbon chain and 1 refers to one double bond) and C18:1. For example, it has been shown previously that expression of medium chain plant thioesterases like FatB from Umbellularia californica in E. coli results in accumulation of high levels of medium chain fatty acids, primarily laurate (C12:0). Similarly, expression of Cuphea palustris FatB1 thioesterase in E. coli led to accumulation of C8-10:0 acyl-ACPs. Similarly, Carthamus tinctorius thioesterase, when expressed in E. coli leads to >50 fold elevation in C18:1 chain termination and release as free fatty acid. The accumulation of fatty acids has been shown to require preventing fatty acid β-oxidation. Mutation of the rate-limiting enzyme governing β-oxidation, acyl-CoA synthase, in E. coli has been shown to greatly improve the production of medium chain fatty acids.
Although the studies described above demonstrated that various fatty acids could be produced in E. coli, there are several disadvantages in using E. coli as opposed to using photosynthetic microorganisms as a production host for fatty acids. First of all, production of any renewable chemical in E. coli requires high cost fermentation using sugar which itself has to come from plant sources. In contrast, photosynthetic microorganisms directly utilize carbon dioxide and sunlight as an energy source and in the case of cyanobacteria, many also utilize atmospheric nitrogen gas as a nitrogen source. There are also several differences in E. coli metabolism that differentiate it from the photosynthetic microorganisms such as cyanobacteria with respect to biosynthesis and utilization of fatty acids. Whereas E. coli requires inactivation of the fatty acid β-oxidation pathway to allow for any significant production of fatty acids, similar measures are not generally required in Cyanobacteria, which appear not to utilize fatty acids as an energy source—thus allowing for high levels of their production. This likely stems not only from the fact that cyanobacteria are generally photoautotrophs (unlike E. coli, which are heterotrophs), but also stem from the profound difference in utilization of exogenous fatty acids by the two organisms. E. coli utilizes exogenous fatty acids as an energy source by coupling them to CoA with the help of acyl-CoA synthetase. The acyl-CoA precursors are then broken down to acetyl-CoA. Cyanobacteria lack this activity; instead, they couple exogenous fatty acids to ACP using acyl-ACP synthase. Those precursors are then gradually incorporated into lipids. Thus, whereas acyl-CoA synthetase activity in E. coli directly competes with a thioesterase by committing the acyl chain to a degradation pathway, the acyl-ACP synthetase in Cyanobacteria simply reverses the thioesterase activity.
In addition to changes in fatty acid utilization, Cyanobacteria biosynthesize only saturated fatty acids (C16:0 and C18:0) as opposed to E. coli, which biosynthesizes both saturated and unsaturated fatty acids. Thus expression solely of a thioesterase in Cyanobacteria results in production of only saturated fatty acids—a more pure feedstock as opposed to a mixture of saturated and unsaturated fatty acids produced by E. coli. To generate unsaturated fatty acids in cyanobacteria, an ACP-desaturase and a thioesterase may be co-expressed. Whereas ACP-desaturases are inactive in E. coli due to lack of ferredoxin, cyanobacteria do not have that limitation.